A New Anaesthetic Protocol for Adult Zebrafish (Danio rerio): Propofol Combined with Lidocaine

Background

The increasing use of zebrafish model has not been accompanied by the evolution of proper anaesthesia for this species in research. The most used anaesthetic in fishes, MS222, may induce aversion, reduction of heart rate, and consequently high mortality, especially during long exposures. Therefore, we aim to explore new anaesthetic protocols to be used in zebrafish by studying the quality of anaesthesia and recovery induced by different concentrations of propofol alone and in combination with different concentrations of lidocaine.

Material and Methods

In experiment A, eighty-three AB zebrafish were randomly assigned to 7 different groups: control, 2.5 (2.5P), 5 (5P) or 7.5 μg/ml (7.5P) of propofol; and 2.5 μg/ml of propofol combined with 50, (P/50L), 100 (P/100L) or 150 μg/ml (P/150L) of lidocaine. Zebrafish were placed in an anaesthetic water bath and time to lose the equilibrium, reflex to touch, reflex to a tail pinch, and respiratory rate were measured. Time to gain equilibrium was also assessed in a clean tank. Five and 24 hours after anaesthesia recovery, zebrafish were evaluated concerning activity and reactivity. Afterwards, in a second phase of experiments (experiment B), the best protocol of the experiment A was compared with a new group of 8 fishes treated with 100 mg/L of MS222 (100M).

Results

In experiment A, only different concentrations of propofol/lidocaine combination induced full anaesthesia in all animals. Thus only these groups were compared with a standard dose of MS222 in experiment B. Propofol/lidocaine induced a quicker loss of equilibrium, and loss of response to light and painful stimuli compared with MS222. However zebrafish treated with MS222 recovered quickly than the ones treated with propofol/lidocaine.

Conclusion

In conclusion, propofol/lidocaine combination and MS222 have advantages in different situations. MS222 is ideal for minor procedures when a quick recovery is important, while propofol/lidocaine is best to induce a quick and complete anaesthesia.     

Gut Hormone Pharmacology of a Novel GPR119 Agonist (GSK1292263), Metformin,and Sitagliptin in Type 2 Diabetes Mellitus: Results from Two Randomized Studies

GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25–800 mg; n = 45) or multiple doses (100–600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ∼five-fold compared with placebo, reaching peak concentrations of ∼50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ∼100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides.

Trial Registration:

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119846","term_id":"NCT01119846"}}NCT01119846 Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01128621","term_id":"NCT01128621"}}NCT01128621     

Non-induced leukocyte extract reduces HIV replication and TNF secretion

According to UNAIDS, the global HIV/AIDS epidemic increased to 40 million the number of people living with the virus around the world. Dialyzable leukocyte extract obtained by our group is a low molecular weight dialyzable material from peripheral human leukocytes previously in vitro induced with Sendai virus (DLE-ind), and more recently, from non-induced leukocytes (DLE n/i). Previous results have shown the ability of DLE-ind to inhibit HIV in vitro replication in MT4 cell; to reduce TNFalpha secretion, and to delay in vivo progression to AIDS in early stage of HIV infection. In this work we present evidences that DLE n/i also inhibits HIV in vitro replication and reduces TNFalpha secretion in human whole blood like DLE obtained from induced leukocytes. Taking together these results show that both properties of DLE, HIV in vitro inhibition and TNF production modulation, are not dependent on in vitro Sendai virus induction of leukocytes.     

The spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns

It is believed that pericentromeric heterochromatin may play a major role in the epigenetic regulation of gene expression. We have previously shown that centromeres in human peripheral blood cells aggregate into distinct "myeloid" and "lymphoid" spatial patterns, suggesting that the three-dimensional organization of centromeric heterochromatin in interphase may be ontogenically determined during hematopoietic differentiation. To investigate this possibility, the spatial patterns of association of different centromeres were analyzed in hematopoietic progenitors and compared with those in early-B and early-T cells, mature B and T lymphocytes, and, additionally, mature granulocytes and monocytes. We show that those patterns change during lymphoid differentiation, with major spatial arrangements taking place at different stages during T and B cell differentiation. Heritable patterns of centromere association are observed, which can occur either at the level of the common lymphoid progenitor, or in early-T or early-B committed cells. A correlation of the observed patterns of centromere association with the gene content of the respective chromosomes further suggests that the variation in the composition of these heterochromatic structures may contribute to the dynamic relocation of genes in different nuclear compartments during cell differentiation, which might have functional implications for cell-stage-specific gene expression.     

Proteomic Characterization of Murid Herpesvirus 4 Extracellular Virions

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi’s sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.     

Siaα2-3Galβ1- Receptor Genetic Variants Are Associated with Influenza A(H1N1)pdm09 Severity

Different host genetic variants may be related to the virulence and transmissibility of pandemic Influenza A(H1N1)pdm09, influencing events such as binding of the virus to the entry receptor on the cell of infected individuals and the host immune response. In the present study, two genetic variants of the ST3GAL1 gene, which encodes the Siaα2-3Galβ1- receptor to which influenza A(H1N1)pdm09 virus binds for entry into the host cell, were investigated in an admixed Brazilian population. First, the six exons encoding the ST3GAL1 gene were sequenced in 68 patients infected with strain A(H1N1)pdm09. In a second phase of the study, the rs113350588 and rs1048479 polymorphisms identified in this sample were genotyped in a sample of 356 subjects from the northern and northeastern regions of Brazil with a diagnosis of pandemic influenza. Functional analysis of the polymorphisms was performed in silico and the influence of these variants on the severity of infection was evaluated. The results suggest that rs113350588 and rs1048479 may alter the function of ST3GAL1 either directly through splicing regulation alteration and/or indirectly through LD with SNP with regulatory function. In the study the rs113350588 and rs1048479 polymorphisms were in linkage disequilibrium in the population studied (D’ = 0.65). The GC haplotype was associated with an increased risk of death in subjects with influenza (OR = 4.632, 95% CI = 2.10;1.21). The AT haplotype was associated with an increased risk of severe disease and death (OR = 1.993, 95% CI = 1.09;3.61 and OR 4.476, 95% CI = 2.37;8.44, respectively). This study demonstrated for the first time the association of ST3GAL1 gene haplotypes on the risk of more severe disease and death in patients infected with Influenza A(H1N1)pdm09 virus.     

Trisomy 8, a Cytogenetic Abnormality in Myelodysplastic Syndromes,Is Constitutional or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS.     

High-level expression and immunogenic properties of the recombinant rabbit hemorrhagic disease virus VP60 capsid protein obtained in Pichia pastoris

The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.     

Biochemical parameters to assess cell differentiation of Bouvardia ternifolia Schlecht callus

Calli derived from leaves and radicles of B. ternifolia were grown on Murashige and Skoog (MS) basal medium, and the effects of different nitrogen sources on the rate of callus growth and on the enzymes related to nitrogen assimilation were studied. Ammonium alone did not support callus growth unless a Krebs-cycle intermediate was added to the medium. The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (EC 1.4.1.2) were measured in homogenates of callus grown on media supplied with different nitrogen sources. The results indicate that leaf and root calli have similar levels of these enzymes when grown on MS medium (Murashige and Skoog 1962. Physiol. Plant. 15, 473–497). However, when the calli were supplied with glutamine as the sole nitrogen source, the activity of glutamate synthase increased in leaf callus but was almost completely inhibited in root callus. The results indicate that calli originated from different B. ternifolia tissues do not have the same biochemical dedifferentiated state.